The ribosome synthesizes proteins in all living organisms. Despite significant progress in the research of protein synthesis, many molecular details of translation are unknown and the study of the ribosome remains one of the biggest challenges in structural biology. Protein synthesis is a dynamic process, during which the ribosome moves along mRNA and tRNAs are translocated inside the ribosome. The long-term goal of the proposed project is to elucidate conformational transitions of the ribosome and ribosomal ligands underlying translation. The proposal is focused on the mechanics of ribosome movement along mRNA and the molecular mechanism of ribosome recycling, i.e., ribosome disassembly into subunits after termination of protein synthesis. Both of these processes in bacteria involve protein factor EF-G (EF-2 in eukaryotes). Molecular details of translocation and recycling are poorly understood. It has been shown that ribosome translocation along mRNA requires ratchet-like rotation between ribosomal subunits and that intersubunit rotation is thermally driven and occurs spontaneously. The Specific Aim 1 of the proposal is to probe mechanistic properties of the ribosome as a Brownian ratchet machine whose spontaneous intersubunit rotation can be rectified into translocation by ligand binding. We will use antibiotic as a tool to examine whether antibiotics whose binding sites overlap with the A site on the large ribosomal subunit can induce ribosome translocation. The Specific Aim 2 will address the question of how the natural catalyst of ribosomal translocation, EF-G rectifies spontaneous intersubunit movements into unidirectional translocation of the ribosome. The extended domain IV of EF-G has been shown to bind to the A site of the post-translocation ribosome. It has been hypothesized that movement of the extended domain IV relative to the A site and the rest of EF-G is critical for translocation. We are going to test this hypothesis by following movements of domain IV of EF-G during translocation using Frster resonance energy transfer (FRET) in single-molecule and stopped-flow kinetic measurements. The experiments of Specific Aim 1 and 2 will elucidate the mechanism of translocation and contribute to understanding of how molecular machines, such as DNA/RNA polymerases and helicases, move along nucleic acids. EF-G is also involved in ribosome recycling that is induced by the concerted action of EF-G and ribosome recycling factor (RRF). The Specific Aim 3 is to study conformational changes of the ribosome, RRF and EF-G associated with ribosome recycling using FRET. Proposed experiments will reveal the sequence of structural transitions during ribosome recycling and help to understand its molecular mechanism. Our studies will illuminate essential steps of protein synthesis and thus answer fundamental biological questions.